The use of combinatorial approaches for protein identification, characterization and modification has been highly successful in both academic and commercial research and development. In this respect, filamentous bacteriophage, or phage, display technology has paved the way being the first library platform and still thrones as the dominating technology. Thus, phage display is widely applied in both basic and applied protein discovery, as well as in development of both novel protein-based diagnostics and therapeutic, which are the class of compounds most rapidly growing world-wide.
The principle of combinatorial phage display technology is based on the genotype—phenotype linkage offered by the property that each virion will only display on its surface the very same proteins that are encoded by the genome encapsulated by its protein coat. The phage particle itself is highly resistant to a variety of physiochemical conditions; hence phage display offers superior versatility in many selection regimes as compared to competing combinatorial technologies.
Phage display of heterologous polypeptides has been achieved using all five structural proteins of the filamentous phage coat, but only pIII- and to some extent pVIII-display have gained widespread use (FIG. 1).
When the heterologous fusion is only a short peptide, multivalent display systems using phage genome-based vectors are preferred, whereas for larger fusions requiring folded domains most applications will benefit from the phagemid systems. In the latter case, antibody-pIII phage display is by far dominating the field, but alternative scaffolds are emerging at dawns early light, continuing the need for expansion of protein engineering tools of tomorrow. For many applications, it would be highly advantageous to be able to make, specifically and in a controlled manner, bispecific phage particles in that more than one of the coat proteins displayed a fusion peptide in the context of the same virus particle. Also, such a system should not interfere with already established display approaches and in particular pIII and pVIII display.
Endemann and Model, 1995 (PMID: 7616570), reported that the minor coat protein pVII was not accessible in the intact phage and that pVII was not functional with another protein fused to its N-terminus. Thus, this report concluded that pVII cannot be used for phage display.
Gao et al, 1999 (PMID: 10339535) and patent application WO0071694, describes heterologous peptide phage display on pVII using the octapeptide FLAG tag, as well as simultaneous phage display on pVII and pIX to generate functional heterodimeric polypeptides harbouring complex folding topologies (antibody Fv). These authors aimed at developing an alternative means for antibody display. The pVII and pIX fusion proteins were expressed from a phagemid employing a dicistronic constellation, hence the resulting functional phage particles inevitably contained varying amounts of pVII and pIX fusion proteins due to complementation by wild type pVII and pIX protein donated from the helper phage genome. As mentioned above, it had previously been suggested that pVII and pIX were not functional with another protein fused to their N termini, and Gao et. al. gave two possible reasons for their success, either alone or by the combination of both.
One possible reason was that a prokaryotic leader sequence (signal sequence) was attached N-terminally to the fusion proteins, thus ensuring targeting of the recombinant protein to the periplasmic space and thereby prevented accumulation in the cytoplasm. Another possible reason was that the recombinant proteins were expressed from a phagemid, not a phage genome as by Endemann and Model, hence wild type pVII and pIX from the helper phage inevitably needed for phagemid rescue were complementing the recombinant pVII and pIX fusion proteins, thus preserving wildtype functionality that otherwise may have been lost due to the recombinant modification. I.e. the phages would comprise a mix of wild-type and fusion proteins. The authors mention that the pVII-pIX display format would be particular useful for combinatorial display of heterodimeric arrays, which, for unknown reasons, appear to yield a particular powerful enrichment during panning protocols. The authors do not envisage using pVII as sole displaying protein (as phagemid or phage genome) or using pVII display in combination with display at another coat protein (different from pIX) to achieve bispecific display.
Kwasnikowski et al. (PMID: 16277988) described genetically stable fusion of scFv fragments to gene VII directly in the phage genome. I.e. the resulting phages comprised no native pVII protein, and the pVII display was multivalent. The authors speculate that one of the reasons for successful pVII display in the phage genome format is that they supported the fusion gene with a prokaryotic signal sequence that directs the fusion protein to the periplasmic space. The authors argued that the unique feature of their system is that the pVII displaying phages bears unmodified, wild-type pIII minor coat protein. Since it has been reported that multiple copies of functional pIII are required for host cell infection, the presence of wild-type pIII of the phage surface may facilitate recovery of selected antibodies with larger diversity. Thus, the authors do not envisage bispecific display, nor do they envisage pVII display without a prokaryotic signal sequence targeting to the periplasmic space.
Khalil et al (PMID: 17360403) describes an application exploiting the feature of a bispecific filamentous phage virion in which an exogenous peptide is displayed at each distal tip of the very same virion. They achieved this by using the combination of a common pIII phage genome vector complementing a pIX display phagemid. In this setting, the phage genome vector served as a helper phage in rescuing the phagemid, thus being reminiscent of the approach described herein of creating a bispecific phagemid virion by rescuing a pIII display phagemid by the use of a pVII modified helper phage genome. Moreover, the bispecific virions of Khalil et al display a peptide-pIII fusion that allows for a controlled biotinylation of their virion. There are however, several features that differ between these two avenues of obtaining a bispecific virion, as well as obtaining defined virion biotinylation, which make them unique from each other.
Firstly, the approach of Khalil et al cannot be used in combination with pIII phagemid display, as it is their phage genome vector that carries their pIII fusion, hence bispecificity cannot be obtained upon phagemid rescue and it would also highly likely be deleterious to the functionality of both pIII fusions.
Secondly, and as the authors also themselves pinpoint, genomic pIX modifications are not regarded as a viable strategy due to overlapping genes in the phage genome, thus they do not envision or speculate in making any modified helper phage genome that can be used for pIII phagemid (or pVIII) rescue and by this way donate a defined phenotypic feature to both distal tips of the very same virion. Khalil et al do never mention the use of modified pVII in either phagemid, or phage genome display.
Thirdly, Khalil et al do not speculate in modifying a single phage genome to achieve a bispesific virion, by exploiting simultaneous modification of more than one capsid gene within the very same genome. They merely use standard pIII peptide display through a commercially available phage genome vector.
Forth, Khalil et al only make bispecific virions displaying short peptides, not folded domains, and do never speculate in exploiting such display at either on, or both modified capsid proteins.
Fifth, Khalil et al achieve site-specific biotinylation of their pIII displayed peptide through in vitro chemical conjugation, not by an enzymatic reaction either in vitro or in vivo. The authors never envision enzyme mediated biotinylation of a displayed moiety by displaying an enzymatic substrate such as AviTag.
Finally, does Khalil et al. not show any type of display without the use of a N-terminal signal sequence.